Diagnosis of human metapneumovirus by immunofluorescence staining with monoclonal antibodies in the North-East of England.

BACKGROUND: Since its discovery in 2001 human metapneumovirus (hMPV) has been shown to be a significant cause of human respiratory disease, responsible for 5-8% of respiratory infections in hospitalised children. Diagnosis hitherto has been largely carried out by reverse tanscriptase polymerase chain reaction (RT-PCR) but immunofluorescence staining of cells from nasopharyngeal secretions (IF) offers advantages for some laboratories and may produce a more rapid result in urgent cases. We have recently demonstrated that IF with a rabbit antiserum gave sensitivity equal to that of RT-PCR. However, monoclonal antibodies offer a more plentiful, uniform IF reagent. OBJECTIVES: Here we have evaluated a pool of anti-hMPV monoclonal antibodies in the routine diagnosis of respiratory infections in hospitalised infants and children. STUDY DESIGN: Eight hundred and fifty-seven routine respiratory specimens were tested by IF with rabbit polyclonal antiserum and monoclonal antibody pool in parallel. A further 1003 specimens were tested with the monoclonal antibody pool alone. All specimens were also tested for a panel of other respiratory viruses by IF. RESULTS: Both rabbit polyclonal antiserum and monoclonal antibody pool gave positive results in 56 and negative results in 797 specimens. The rabbit polyclonal antibody detected virus in a further two specimens which were negative when tested with the monoclonal pool giving a concordance of 96.6% and a specificity of 100% for the monoclonal antibody pool. Overall hMPV was detected in 5% of specimens whilst 18.4% were positive for hRSV. CONCLUSIONS: The monoclonal antibody pool-based IF is a robust assay suitable for routine use with a sensitivity only slightly less than that of the other major diagnostic methodologies available.

Detection of human metapneumovirus in respiratory secretions by reverse-transcriptase polymerase chain reaction, indirect immunofluorescence, and virus isolation in human bronchial epithelial cells.

Over two winters in Newcastle upon Tyne, respiratory secretions, negative by immunofluorescence staining for other respiratory viruses, were tested for the presence of human metapneumovirus (HMPV) by RT-PCR. In the second winter, specimens were also tested by immunofluorescence staining with an anti-HMPV polyclonal rabbit antiserum and immunofluorescence positive specimens were inoculated into a line of human bronchiolar cells, 16HBE140. Overall, 55 of 549 (10%) specimens tested were positive for HMPV by RT-PCR. Of 162 specimens tested by both RT/PCR and immunofluorescence staining, 23 were positive by both techniques. Of five specimens positive by RT-PCR alone, only one was confirmed with a second set of primers. Of three specimens positive by immunofluorescence alone, only one was confirmed by virus culture. All four previously recognized sub-genotypes of the virus were identified by both RT-PCR and immunofluorescence staining. Sub-genotype A1 was prevalent in the first winter and B1 prevalent in the second. HMPV replication and virus isolation rates were higher in 16HBE140 cells than in monkey kidney cells and did not require exogenous trypsin. Low passage isolates of both sub-genotypes A2 and B1 replicated slowly reaching peak titers only 12 days after inoculation. In summary, single round RT/PCR and immunofluorescence staining with a polyclonal rabbit antiserum proved of equal sensitivity in the diagnosis of HMPV infection in respiratory secretions both detecting 96% of confirmed positive specimens. 16HBE40 cells provided a significant improvement on monkey kidney cells for the isolation and propagation of the virus.

Bond strengths of dentinal bonding systems to enamel and dentin.

Contemporary, third-generation dentinal bonding products have become highly specialized in producing high bond strengths to dentin. This investigation compared in vitro bond strengths of six dentinal bonding systems and their matched composite resins to human enamel and dentin. The effects of treatment by dentinal primers on enamel bond strengths as well as the effects of phosphoric acid on the strengths of dentinal bonds were measured. The use of dentinal primer on enamel improved the bond strengths of Prisma Universal Bond 3/Prisma APH and XR Bond/Herculite systems and had no effect on Denthesive/Charisma, Scotchbond 2/Silux, and Tenure/Perfection, while the enamel bond strengths of Gluma/Pekalux declined. Pretreatment of dentin with phosphoric acid improved the bond strengths of Denthesive/Charisma, Prisma Universal Bond 3/Prisma APH and XR Bond/Herculite, but had no effect on Gluma/Pekalux, Scotchbond 2/Silux and Tenure/Perfection.

Diagnosis and treatment of an oral base-metal contact lesion following negative dermatologic patch tests.

We report a confirmed case of intraoral contact mucositis secondary to nickel dental alloy hypersensitivity. The lesion resolved after removal of the offending prosthesis. The patient responded negatively to dermatologic patch tests, but a positive intraoral rechallenge confirmed the mucositis diagnosis. A nonreactive, gold alloy prosthesis was inserted for a successful result.

In vitro bond strength of two adhesives to enamel and dentin under normal and contaminated conditions.

In vitro bond strengths of human enamel and dentin treated with five contaminants were measured with air, water and damp conditions as controls. Two commercial bonding agents (a lower-viscosity, solvent-containing type, AB, and a higher-viscosity, hydrophilic monomer type, SB) and their composites were applied to tooth structure under two conditions (contaminated and re-etched). Samples were debonded in tension after 24 h using an inverted, truncated cone test. Among the controls, the highest bond strengths were obtained with damp conditions for AB (24 MPa) and damp conditions or air for SB (22 MPa) with small differences between enamel and dentin. Most contaminants lowered the bond strength. Re-etching without additional mechanical preparation resulted in bond strengths similar to controls. Bond strengths to tooth structure with the bonding agents tested may be less sensitive to common forms of contamination than typically assumed.

Determination of the existence of hinge movements of the temporomandibular joint during normal opening by Cine-MRI and computer digital addition.

PURPOSE: The purpose of the first phase of this two-part investigation was to determine if the opening motion of the mandible could be illustrated and described using a dynamic imaging method. The purpose of the second phase of the investigation was to determine if a center of rotation would be discovered. MATERIALS AND METHODS: Five volunteer subjects, 2 female and 3 male, whose temporomandibular joints had previously been determined to be asymptomatic, were examined by magnetic resonance imaging (MRI) during opening from a standardized position. The serial static images were reconstructed by the MRI's computer in "cine mode" to simulate dynamic motion, similar to a motion picture. For the second phase, each patient's series of static images were digitally added and manipulated by a computer graphics program to locate the center of hinge motion. RESULTS: After reviewing the animated images recorded on videotape, three independent dentist observers confirmed that the opening movement of the mandible was initially rotational, followed by translation within the glenoid fossa. A center of rotation was calculated to be in the anatomic center of the condylar head of all of the subjects in this study. CONCLUSIONS: This study showed that opening dynamics of the mandibular condyle could be studied by cine-MRI and that an opening hinge axis appears to be located in the anatomic center of the condylar head.

Alterations in human enamel surface morphology following vital bleaching.

Extracted intact human teeth (n = 4) were treated for 30 days by three protocols: Home 1 (Proxigel, n = 4) for 8 hours daily, Home 2 (White & Bright, n = 4) for 24 hours with 3 minutes of stannous fluoride gel, or an Office protocol (n = 4) using 30% hydrogen peroxide (Superoxol) warmed by a high-intensity light while the controls remained untreated. "Home" bleaching agents contain approximately 10% carbamide peroxide. After treatment, the coronal surfaces were examined with a scanning electron microscope (SEM) at 2000 power magnification, and the surface topography was measured by a profilometer. The SEM photomicrographs of the controls and office-treated groups were similar to previously reported descriptions, while the home bleached surfaces appeared similar to each other. Profilometric analysis was used to examine surface roughness and surface waviness. Mean surface roughness in microns was: control, 1.9; Home 1, 0.6; Home 2, 0.9; and Office, 0.6. Surface waviness was ranked control > office > Home 1 = Home 2. Enamel surface alterations were evident after the three bleaching methods. The differences between the office and home-treated surfaces were unrelated to the pH of the bleaching agents.

Enamel shear bond strengths after vital bleaching.

This study examined the shear bond strengths of enamel bond formed at specific time intervals after the termination of vital bleaching. A total of 160 extracted human anterior teeth were divided into three bleaching treatment (BTx) groups (Superoxol, Proxigel, White & Brite), two bonding agent (BA) subgroups (Scotchbond Dual Cure, Scotchbond 2) and two BA control groups. After BTx for 30 days, bonds were formed at 1, 6 and 24 hours; 3 and 7 days (time interval, TI) post-termination of BTx. A total of 32 groups of n = 5 were used. Bonds were formed in nylon tubes with selected BA and Silux Plus, and ruptured 24 hours later with Instron and cable/loop to measure enamel shear bond strength (SBS). A 3 x 2 x 5 factorial ANOVA and Tukey 95% confidence interval (TCI) detected significant differences between BTx (F = 3.54; TCI = 2.7 MPa), BA (F = 9.73; TCI = 1.8 MPa) and TI (F = 9.39; TCI = 4.1 MPa). The interaction between BTx and TI was slightly significant (F = 2.02); other interactions were not significant. Post-hoc analysis showed 80% of bonds failed at the interface or by mixed cohesive composite/interfacial failure. Scanning electron microscope observations suggested association of increased density of voids in bond area at TI with lowest mean bond strengths. SBS ranged from 42.3 to 106.8% of control.

In vitro bond strength of silica-coated metal posts in roots of teeth.

This study determined the in-vitro bond strength of abrasive-sprayed and silica-coated Ni-Cr-Be posts to roots of extracted teeth using three resin cements (Panavia EX [P], Super-Bond C&B [SB], Prisma Universal Bond 2-Dicor [PUB-D]) and zinc phosphate cement (ZP). There were no significant differences among bond strengths of resin cements (8.8 to 10.8 MPa) bonded to abrasive-sprayed posts, but the bonds were stronger than those obtained using zinc phosphate cement (4.4 MPa). With the silica-coated posts, Super-Bond C&B produced the highest bond strength (14.5 MPa), followed by PUB-D (10.9 MPa), P (7.5 MPa), and ZP (5.4 MPa).

Shear bond strength of Scotchbond in vivo.

The shear bond strengths of Scotchbond, HEMA/Scotchbond, and Scotchbond 2 were measured in vivo in dog canine and molar enamel and dentin. Dentin bond strengths were compared in superficial, middle, and deep dentin. The acid-etched enamel bond strengths of the three bonding systems ranged from 10 to 11 MPa and were not statistically different. Scotchbond/Silux bonds to superficial and middle cuspid and molar dentin were 3 MPa and were not statistically different. HEMA-treated dentin did not consistently improve Scotchbond strengths to either tooth type at any dentin depth. Deep dentin from either tooth type yielded significantly lower bond strengths. Scotchbond 2/Silux shear bond strengths were significantly higher (6-8 MPa) in superficial and middle cuspid dentin but were not different from Scotchbond bonds made to deep cuspid dentin or to any depth of molar dentin. The observation that molar bond strengths are lower than those made to cuspid dentin indicates that there are important substrate differences between teeth as well as within dentin as a function of depth. The dog model may be useful for the screening of new dentin bonding systems prior to clinical trials.

Effect of disinfection/sterilization treatments on Gluma-mediated dentin shear bond strengths.

This in vitro study investigated Gluma-mediated dentin shear bond strengths after treatment by three commonly used disinfection/sterilization methods: 1) proprietary phenolated glutaraldehyde (Sporicidin); 2) 1% NaOCl and; 3) autoclaving. Varying numbers of bonds were too low to be measured, failing prior to, or early in testing. Failed bonds were scored as zero. When all attempted bonds were subjected to ANOVA, there was a significant difference between groups (P less than 0.0001) in bond strengths. These results demonstrate the significant differences were due to statistical management; that is, inclusion of zeros. When only measurable bonds were similarly analyzed, no significant differences in bond strength were found. When the measurable bonds were tested by Fisher's exact test, significant differences were detected among and between the experimental groups. Additionally, the experimental treatments probably altered or removed dentinal protein constituents. A plea is made for clarity and uniformity in statistical management and reporting of bonds which are too low to be measured.

Observations on clinical and immunofluorescent diagnosis of parainfluenza virus infections.

Immunofluorescent techniques have been applied to nasopharyngeal secretions for the rapid diagnosis of parainfluenza virus types 1, 2, and 3 infections. Seventy-five infections were found by isolation techniques; 55 of these had nasopharyngeal secretions taken and 53 were positive by direct examination. A comparison of the results of 60 neutralization tests with immunofluorescence applied to monkey kidney isolations showed complete agreement. Immunofluorescence appeared to be a satisfactory method for differentiating the various haemadsorption viruses. The importance of parainfluenza viruses and respiratory syncytial virus in croup was noted and the association of the parainfluenza viruses with acute respiratory virus infection was confirmed. The clinical relationship between respiratory syncytial virus and parainfluenza virus type 3 is discussed.

The late detection of respiratory syncytial virus in cells of respiratory tract by immunofluorescence.

Paired nasopharyngeal secretions were studied in 27 infants infected with respiratory syncytial virus, one taken at onset of illness and one about 7 days later. Both specimens were examined by immunofluorescence and tissue culture for respiratory syncytial virus. In 25 out of 27 (93%) specific fluorescence was still present in cells of the convalescent specimen but was much duller. Virus was difficult to isolate in convalescent specimens; only 8 out of 27 (26%) proved to be positive. Eight single secretions which were taken late in a respiratory illness were also shown to have this altered fluorescence with absence of virus isolation. Preliminary experiments using antihuman globulin suggest that the findings may be due to the attachment of local secretory antibody to the cells causing ;blocking' of staining reaction.